Method of sample preparation for maldi and automated system therefor

ABSTRACT

Methods for preparing a biological sample for testing by Maldi where such methods are selected based on sample parameters. Maldi scores are obtained for a range of sample parameters (e.g. McFarland, dispense volume and number of dispenses). From the data, sample preparation parameters can be selected for a biological sample being prepared for Maldi testing. One sample preparation strategy uses multiple dispenses of sample with an intervening drying step, which yields more accurate Maldi scores, particularly for samples at the low range of McFarland values (e.g. below about 2).

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/504,549, filed on Feb. 16, 2017, which application is a nationalphase entry under 35 U.S.C. § 371 of International Application No.PCT/US2015/045506 filed Aug. 17, 2015, published in English, whichclaims the benefit of the filing date of U.S. Provisional ApplicationNo. 62/038,509, filed Aug. 18, 2014, all of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

As a routine practice in medical diagnosis and treatment, biologicalsamples such a blood or urine are obtained from a patient and analyzedfor the presence of microorganisms. If microorganisms are determined tobe present, there is both medical and economic justification to bothidentify the specific microorganism present and, to facilitatetreatment, the antibiotic resistance/susceptibility of themicroorganism. Often such determinations must be made quickly to ensurethat the correct treatment is initiated as quickly as possible.

For example, sepsis is a serious medical condition caused by anoverwhelming response of the host immune system to infection. It cantrigger widespread inflammation, which can give rise to impaired bloodflow. As sepsis progresses, the body's organs can be starved for oxygenand nutrients, causing permanent damage and eventual failure. Leftimproperly diagnosed or otherwise untreated, the heart weakens andseptic shock can occur, leading to multiple organ failure and death.Blood cultures are required to detect the presence of bacteria or yeastin the blood of sepsis patients, to identify the microorganism(s)present and guide treatment. The conventional separation andidentification of microorganism(s) from blood cultures takes at least24-48 hours, which results in many of the septicemia patients beinginitially treated with inappropriate antibiotics. It is thereforedesirable to separate and identify microorganisms from a positiveculture (blood, cerebrospinal fluid etc.) rapidly.

Recently, certain proteomic technologies/tools, such as Matrix-AssistedLaser Desorption Ionization Time of Flight mass spectrometry,(“MALDI-TOF MS”) (“Maldi” hereinafter), have been shown to provide arapid and accurate identification of bacteria and/or fungi from apositive blood culture (“PBC”) or from a bacterial colony grown on asubstrate such as an agar plate.

In the Maldi process, small quantities of microbes from a colonycultivated in the usual way in a nutrient medium are transferred to amass spectrometric sample support plate known as a Maldi plate, and thensubjected directly to mass spectrometric analysis, generally bytime-of-flight (TOF). The mass spectrum analysis shows the differentproteins, provided they are present in the microbes in sufficientconcentration. The identity of the microbe is then determined from themicrobe's protein profile through a computerized search of spectrallibraries containing thousands of reference spectra. If no referencemass spectrum is present in a library for the precise species of microbebeing examined, computerized library searches with looser similarityrequirements can provide at least some indication of the order, familyor genus of the microbes, since related microbes frequently contain anumber of identical protein types. The Maldi process is described infurther detail in International Publication No. WO-2009/065580A1 toUlrich Weller entitled “Identification of Pathogens in Bodily Fluids,”the content of which is hereby incorporated in its entirety. A varietyof mass spectrometry instruments may be used for identification.

The microorganism in the PBC sample can be sub-cultured prior to Maldiidentification, e.g. on an agar plate. In the alternative,microorganisms can be isolated from the PBC sample using various samplepreparation methods without the need for subculturing. The microorganismisolates are generally directly smeared onto a Maldi plate to yieldabout 70-80% identification accuracy. For isolates failing to yield anyidentification, a follow-up liquid extraction method is typically usedto extract proteins from the microorganism for improved identificationby MALDI-TOF MS. Although these liquid protein extraction methodsgenerally yield better identification accuracy, such methods not onlyrequire several centrifugation steps, but also are time-consuming.

Schmidt, V. et al. “Rapid identification of bacteria in positive bloodculture by matrix-assisted laser desorption ionization time-of-flightmass spectrometry,” Eur. J. Clin. Microbiol. Infect Dis. Vol. 31(3), pp.311-317 (March 2012) (Epub dated Jun. 23, 2011) discloses a method ofidentifying bacteria from positive blood cultures by spotting a liquidsample of the isolated bacteria onto a Maldi plate and overlaying 25%formic acid directly to the spotted liquid sample. Therefore, the finalconcentration of formic acid in the bacterial sample is less than 25%.The Schmidt method results in 86.6% identification accuracy forgram-negative bacteria and 60% identification accuracy for gram-positivebacteria. Schmidt did report testing this method in Yeast.

Hyman, J. et al. (U.S. Patent Publication No. 2010/0120085, PublishedMay 13, 2010), discloses a similar method as Schmidt, in which intactisolated microorganisms in solution are directly smeared onto a Maldiplate. The liquid sample is then overlaid with roughly an equal volumeof 50% formic acid. Therefore, the final concentration of formic acidadded to the sample is approximately 25%. This method was tested on 14different species of bacteria and yeast. Although this method resultedin 91.1% identification, the data does not indicate how effective thismethod is with regard to gram-positive bacteria, gram-negative bacteria,or yeast.

Haigh et al. “Improved Performance of Bacterium and Yeast Identificationby a Commercial Matrix-Assisted Laser Desorption Ionisation-Time ofFlight Mass Spectrometry System in the Clinical MicrobiologyLaboratory,” J. Clin. Microbiol. Vol 49(9) p. 3441 (September 2011)describes a method in which neat formic acid is used to extractmicrobial proteins smeared directly onto a Maldi plate. This method,however, was unable to successfully identify all strains of yeast andgram-positive bacteria.

Herendael et al. “Validation of a modified algorithm for theidentification of yeast isolates using matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)”,Eur. J. Clin. Microbiol. Infect Dis Vol 31(5), pp. 841-848 (May 2012)(Epub Aug. 23 2011) describes two methods for the identification ofyeast. The standard extraction method described in Herendael et al., isa conventional liquid extraction method. In the short extraction methoddescribed in Herendael et al., one colony was picked from an agar plateand applied directly to the target Maldi plate. Formic acid (1 μL at 70%concentration) was added to the sample and the sample was allowed todry. The dried sample was overlaid with Maldi matrix, allowed to dryfurther, and analyzed by MALDI-MS. The short extraction method providedidentical results as the standard extraction method although the Maldiscores were lower with the short extraction method. Nearly all of theisolates (97.6%) could be identified with the short extraction method;however 17.1% of these identifications fell below the reliable thresholdlevel of 1.7.

While the extraction method provides accurate results, time to detection(TTD) is much less than detection by direct smear. Therefore, methodsthat improve the accuracy of identification using direct smear Maldi aresought.

BRIEF SUMMARY OF THE INVENTION

Various embodiments of the disclosed method enable direct identificationof microorganisms from positive blood cultures (“PBC”) or pure isolatesof bacterial colonies cultured on a substrate by mass spectrometry usingMaldi. In one embodiment of the present invention, sample is preparedfor Maldi using a solution dispense/layering method. In the solutiondispense/layering method described herein, the bacterial suspension thatwill be dispensed is first evaluated to determine its turbidity as anindication of the concentration of bacteria in the suspension. One suchstandard method for the measure of turbidity is the McFarland turbiditystandard. The McFarland turbidity standard is well known to thoseskilled in the art and not described in detail herein.

The bacterial suspension may be created using a method such as themethod described WO2013147610 to Botma et al., entitled “AutomatedSelection of Microorganisms and Identification Using Maldi” and USPatent Publication 2012/0009558 to Armstrong et al. entitled “Method andApparatus for Identification of Bacteria,” the disclosures of which arehereby incorporated by reference in their entirety.

The solution dispense layering method requires, as implied by its name,the formation of two or more layers of solution for Maldi. A selectedvolume of sample is applied on the Maldi plate and dried. Subsequently,at least a second layer of sample is applied (preferably the samevolume) as the first layer. The second layer is dried. Optionally, morelayers can be deposited and dried. After the final of the two or morelayers is dried, the sample is processed for Maldi (e.g. by addingformic acid and then applying the matrix over the sample as describedherein). The sample is then evaluated by Maldi. The solutiondispense/layer method has been determined to provide acceptable Maldiresults for liquid samples with McFarland turbidity values significantlyless than 2.0 for both Gram positive and Gram negative bacteria.

For example, in one embodiment, if the liquid bacterial suspension(prepared from a bacterial colony picked from an agar plate andsuspended in water (mass spectrometry grade) as referenced above, has avalue of 0.5 McFarland, that value is significantly below the value of2.0 McFarland, which is an indication that the solutiondispense/layering sample preparation should be used to prepare thissample for Maldi.

After determining to use solution dispense/layering to prepare thesample for Maldi, the amount of sample is selected per layer. In theabove example with a sample having a 0.5 McFarland value, the volume perlayer of at least about 3 μl but not exceeding about 4 μl is selected.The number of layers is governed by the turbidity value and the samplevolume. Once the volume of the layer is selected and deposited on theMaldi plate, the sample is dried. The exact drying conditions are amatter of design choice and are selected to provide quick drying whilepreserving sample integrity for Maldi testing. Suitable dryingconditions are readily determined by one skilled in the art. Forexample, the drying steps can be completed at either ambient temperatureor with the assistance of a hot plate (illustratively, about 40° C. toabout 45° C.). After drying, a second layer of sample is deposited overthe first layer. The second layer has the same volume as the firstlayer. If needed, additional layers are added and dried. Since thelayering method requires additional time and resources, the number oflayers is limited to that number needed to obtain accurate results fromMaldi.

Following the solution dispense/layering sample deposition, the sampletarget well is processed using the typical Maldi procedure (addition of70% formic acid and matrix).

It has been determined that the sample preparation process for Maldidepends upon a variety of factors, but most significantly: i) theconcentration of the microorganisms in the sample; ii) the volume of thesample; and iii) if applicable, the number of dispenses. The microbialconcentration is reflected by the turbidity of the sample. Roughly, thehigher the turbidity, the higher the microbial concentration. The methoddescribed herein begins with a sample turbidity measurement. Turbidityis measured by standard nephelometry using techniques and equipment wellknown to the skilled person. Nephelometry is not described in detailherein. Once the turbidity is assessed, a decision on how to go aboutsample preparation for Maldi is made. Such a determination is made byevaluating the turbidity information and sample volume. In thoseembodiments of the present invention where an automated evaluation anddetermination is contemplated, the sample information is entered into adata base. The data base (pre-programmed with information regarding thesample preparation best suited to the particular sample) outputs therecommended method for Maldi sample preparation.

In another embodiment, a system for evaluating a sample and determiningthe appropriate Maldi preparation protocol is contemplated. Preferably,the system is fully automated. In the automated embodiment of thesystem, a processor controls the Maldi preparation protocol, dependingupon information that the processor receives regarding the sample. Thesample is obtained either directly from a PBC or picked from a platedculture. In the automated systems, the sample is obtained usingrobotics. Robotic mechanisms that obtain biological samples for testingare well known to the skilled person and not described in detail herein.In other embodiments, the system is semi-automated. In thesemi-automated embodiments, the sample is obtained manually.

After the sample is obtained, it is diluted. In the automated system,dilution is controlled by instructions from the processor. The systemdilutes the sample to a predetermined volume (e.g., 4.5 ml) usingsterile water as described above. Dilution can also be performedmanually in the semi-automated embodiments.

Once the sample is diluted, the system measures sample turbidity.Preferably, the system deploys automated equipment to measure turbidity.Automated equipment for measuring sample turbidity is known to oneskilled in the art and not described in detail herein. In thesemi-automated embodiments, turbidity is measure manually.

The system processor compares the measured turbidity with apredetermined turbidity threshold. If the processor determines that thesample turbidity is within a predetermined range of turbidity values,then the processor provides instructions to transfer a predeterminedvolume of the diluted sample to the Maldi plate. The automated systemprepares the sample for Maldi (i.e. the addition of formic acid todisrupt the cell wall of the microorganisms thereby releasing theirproteins followed by application of Maldi matrix solution over thesample prior to Maldi as described elsewhere herein) based oninstructions from the processor. In the semi-automated system, onceinstructions for the volume of sample to be deposited on the Maldi plateare received, the Maldi plate is prepared manually. If the processordetermines that turbidity is above the predetermined range, theprocessor provides instructions to prepare a Maldi sample using lessthan the typical volume (i.e. if a 0.5 μl is normally deposited on theMaldi plate, only 0.25 μl is deposited on the Maldi plate instead forthe high turbidity samples). If the processor determines that turbidityis below the predetermined range, the sample is deposited in layers onthe Maldi plate, with drying of the sample between deposits. Thedeposition of multiple layers of the sample on a Maldi plate isdescribed above. As noted above, in the fully automated embodiments, thesystem has automation that deposits the sample on the Maldi plate basedon instructions from the processor. In the semi-automated embodiment, anoperator deposits the sample on the Maldi plate based on instructionsfrom the processor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of data used to formulate a statistical modelused in one embodiment of the method described herein, including theparameters used and the values of the parameters used;

FIG. 2 illustrates additional conditions used to develop the statisticalmodel described herein;

FIGS. 3A and 3B are lists of microorganisms that were used to test themethod describe herein;

FIGS. 4A-4C are surface response plots from statistical models thatillustrate what Maldi sample preparation parameters yield acceptableMaldi results;

FIG. 5 describe the number of correct identifications (of the bacterialisted in FIG. 3) from Maldi (species identification) for differentsample preparation parameters;

FIGS. 6A and 6B compares the Maldi score for 0.25 McFarland samplesprepared with multiple dispenses with and without drying, for a varietyof different microorganisms;

FIG. 7A-7C compares the Maldi score for 0.25 McFarland samples preparedwith different sample volumes/number of dispenses, for a variety ofdifferent microorganisms;

FIG. 8 is a flow chart for the embodiment of the present invention wheresample preparation is based upon measured sample turbidity; and

FIG. 9 is a schematic of a system used to practice the method describedherein.

DETAILED DESCRIPTION

The present invention contemplates methods for preparing samples forMaldi using a solution dispense/layering technique in which multiplelayers of sample are dispensed on a Maldi plate. The dispensed sample isdried between dispenses. Such a method is illustrated herein to offerimprovement in Maldi scores for a broad range of microorganisms.

One skilled in the art is aware that Maldi identification results areaffected by the amount of cells deposited onto the Maldi plate. Astandardized microbial suspension provides a uniform dispersion ofcells, leading to more precise and reproducible results. In oneembodiment, the microbial suspension is standardized prior to depositiononto the solid surface adapted to be placed in an apparatus configuredto determine the identity of microorganisms by Maldi mass spectrometry.The microbial suspension can be optionally adjusted to a certainMcFarland standard. Creation of the standardized microbial suspensioncan be accomplished by various methods well known to those skilled inthe art, for example, using an inoculation loop, micro dropper, or otherphysical methods. In a preferred embodiment, a microbial suspension isadjusted to a McFarland standard of at least 0.25 prior to itsinoculation onto a Maldi plate. The conditions for inoculation (i.e.dispense volume and number of dispenses) are selected to provide asample likely to yield an acceptably high Maldi score, which ispreferably at least about 2 or above.

In the described embodiments, samples for the microorganisms listed inFIG. 3 were prepared for Maldi. Several different methods that were usedto prepare samples for Maldi are described below.

Method 1:

First, a 2.0 McFarland for each microorganism listed in FIG. 3A in asterile diluent (e.g. BBL™ (Becton Dickinson)) was prepared. Three spotswere formed on the Maldi target plate. Each spot was 1 μl in volume. Thesamples were then dried using a hot plate at about 40° C. to about 45°C. The Maldi target plate was obtained from Bruker. After the sampleswere dried, 1 μl of 70% formic acid was applied on the spot. The sampleswere again dried. Then 1 μl of a-cyano-hydroxy cinnanic acid (HCCA)matrix was placed over the spot and the sample was dried and analyzed onBruker Maldi using instrument standard settings. Maldi successfullyidentified the genus and species of 28 out of 29 of the microorganismslisted in FIG. 3A.

Method 2:

First, a 1.0 McFarland for each microorganism listed in FIG. 3A wasprepared using a sterile diluent to dilute the sample. Three spots wereformed on the Maldi target plate. Each spot was 2 μl in volume. Thesamples were then dried using a hot plate at about 40° C. to about 45°C. The Maldi target plate was obtained from Bruker. After the sampleswere dried, 1μl of 70% formic acid was applied on the spot. The sampleswere again dried. Then 1 μl of HCCA matrix was placed over the spot andthe sample was dried and analyzed on Bruker Maldi using instrumentstandard settings. Maldi successfully identified the genus and speciesof 26 out of 29 of the microorganisms listed in FIG. 3A.

Method 3:

First a 1.0 McFarland for each microorganism listed in FIG. 3A wasprepared using a sterile diluent. Three spots were formed on the Malditarget plate. Each spot was 1 μl in volume. The samples were then driedusing a hot plate at about 40° C. to about 45° C. Another 1 μl wasformed on each of the dried spots. Those spots were then dried. TheMaldi target plate was obtained from Bruker. After the samples weredried, 1 μl of 70% formic acid was applied on the spot. The samples wereagain dried. Then 1 μl of HCCA matrix was placed over the spot and thesample was dried and analyzed on a Bruker Maldi using standard settings.Maldi successfully identified the genus and species of 29 out of 29 ofthe microorganisms listed in FIG. 3A.

EXAMPLE 1

Samples with a 0.5 McFarland value were prepared and evaluated for thenumber of microorganisms correctly identified by Maldi. First 0.5McFarland samples for each microorganism listed in FIG. 3A were preparedusing a sterile diluent. The samples were formed on the Maldi plateaccording to the following Table 1:

TABLE 1 Dispense Positive Identifications McFarland Volume Number ofDispenses (out of a total of 29) 0.5 1 μl 1  7 of 29 0.5 1 μl 2 (withdrying between 28 of 29 dispenses) 0.5 1 μl 3 (with drying between 29 of29 dispenses) 0.5 1 μl 4 (with drying between 29 of 29 dispenses) 2 1 μl1 28 of 29

To create the statistical model, four values for each of the threeparameters were selected. The four values and the parameters associatedwith those values are set forth in Table 200 in FIG. 1. These valueswere then loaded into Minitab 16 utilizing the Design of Experiment(DOE) Taguchi Design (which is commercially available, well known to oneskilled in the art and not described in detail herein). This initialdesign created a series of 16 experimental runs using the parametermatrix as set forth in Table 100 of FIG. 1. Table 100 enumerates theinteger associated with the parameters in Table 200. Table 300enumerates the actual parameters for each run. The runs were conductedon the fifteen (15) most difficult bacteria to detect, using Maldiidentification from the challenge set in FIG. 3B (a more challengingarray of microorganisms to detect than those enumerated in FIG. 3A).Data was compiled and analyzed using Statistica version 12 (commerciallyavailable software well known to one skilled in the art). All threeparameters, McFarland, dispense volumes and number of dispenses weresignificant to a determination of whether or not a sample had anacceptable chance to yield a correct Maldi score.

Surface response plots were created and the interactions studied. Thesesurface response plots illustrate that acceptable Maldi results willvary depending upon the values for McFarland, number of dispenses andvolume. The plots illustrate that a higher McFarland is not always thekey to a desirable Maldi score (2.0 or higher for genus and speciesconfirmation). For example a 2.0 McFarland and a lower McFarland (e.g.0.5), could give the same correct Maldi result with a higher dispensevolume (FIG. 4B) and/or a greater number of dispense/dry cycles (FIG.4C) to the target plate. Table 500 sets forth additional sampleparameters for Maldi sample preparation that were used to supplement themodel. These samples had significantly higher McFarland values (˜5.0) todetermine the upper limit interactions.

Maldi values for the samples were obtained using a Bruker MALDI BiotyperLT. All samples were prepared using the above protocols. Values forMcFarland, dispense volume and number of dispenses are as set forth inFIGS. 1 and 2. The runs 1-22 set forth in FIG. 5 were performed for eachmicroorganism enumerated in FIG. 3B. When sample depositions werecomplete, the sample was dried and set with 1 μl of 70 percent aqueousFormic Acid. The sample was again dried and sealed with 1 μl Bruker HCCAMatrix.

To prepare samples to obtain the desired McFarland value the sample wasdiluted with 5 ml of sterile sample diluent. The desired McFarlandvalues were achieved using a BD Phoenix™ Spec Nephelometer and BDPhoenix™ Spec Calibration Kit Methods.

As set forth in FIG. 1, Table 200, the samples were prepared using thefollowing values:

i) McFarland values: 0.25, 0.5, 1.0, 2.0;

ii) Sample volume: 0.5 μl, 1.0 μl, 2.0 μl, 4.0 μl; and

iii) Number of dispenses: 1, 2, 3, and 4.

When sample depositions were complete, the sample was dried and set with1 μl of 70 percent aqueous Formic Acid. The sample was again dried andsealed with 1 μl Bruker HCCA Matrix. Matrix solutions were preparedusing 50% Acetonitrile, 47.5% Water & 2.5% TFA. The solvent was obtainedfrom Sigma Aldrich.

Since three spots were prepared on each plate for each microorganism,three Maldi scores were obtained for each microorganism. So for the 22runs enumerated in Tables 300 and 500 (FIGS. 1 and 2), 66 Maldi scoreswere obtained for each microorganism.

As can be seen from FIG. 5, the highest percentages of correct Maldiscores (a correct result was a Maldi score of 2.0 or above) wereobtained for a variety of different parameters. Generally, for a lowerMcFarland value sample, the volume and number of dispenses is requiredto be higher to get a higher percentage of correct Maldi scores. Forexample, for a 0.25 McFarland sample, a dispense volume of 4 μl and 4dispenses with drying between dispenses was required to achieve 84%correct Maldi scores.

EXAMPLE 2

Samples (0.25 McFarland) were prepared and spiked with themicroorganisms enumerated in FIG. 3B. Samples were prepared using theprocedure described generally above. Three spots were formed on theMaldi target plate. Each spot was 1 μl in volume. The samples were thendried on a hot plate at 40° C. to 45° C. Two target plates were preparedfor each sample. The Maldi target plate was obtained from Bruker. On thefirst plate, the deposited sample was dried, after which another 1 μlspot of sample was placed on the dried first spot. On the second platethe first spot was not dried before the second spot was depositedthereon. After the spots were completely deposited, the spots on bothplates were dried. Then, 1 μl of 70% formic acid was applied on thedried spots on both plates. The samples were again dried. Then 1 μl ofHCCA matrix was placed over the spot and the sample was dried andanalyzed on Bruker Maldi. Maldi successfully identified 39 out of 45spots of spiked samples (at the genus level) for those samples preparedusing a drying step between depositing spots on the Maldi plate. Maldisuccessfully identified 29 out of 45 spots of spiked samples (at thespecies level) for those samples prepared using a drying step betweendepositing spots on the Maldi plate. Since samples were spiked with oneof 15 microorganisms and there were three spots for each spiked sample,a total of 45 sample spots were evaluated by Maldi.

Maldi successfully identified 39 out of 45 spots of spiked samples (atthe genus level) for those samples prepared without a drying stepbetween depositing spots on the Maldi plate. However, Maldi-TOFsuccessfully identified only 24 out of 45 spots of spiked samples (atthe species level) for those samples prepared without a drying stepbetween depositing spots on the Maldi plate. A summary of these resultsis set forth in Table 2 below. Results are reported as the best of thethree, rather than the aggregate of positives. From this it can be seenthat one more microorganism was detected with a drying step betweendeposits than without a drying step.

TABLE 2 0.25 McFarland 1 μl/1 μl (Drying) 1 μl/1 μl (No Drying) Genusand Species ID 29 24 Genus ID only 10 15 No ID  6  6 Correct ID 13/1512/15

The Maldi scores for each microorganism evaluated are listed in FIG. 6.Note that Maldi scores below 1.7 were considered “no identification.”Maldi scores between 1.7 but below 2 were considered positiveidentifications at the genus level but not the species level. Maldiscores at or above 2 were considered positive identifications at bothgenus and species levels. For the samples that were dried between firstand second dispenses, the percent of positive identifications at thegenus and species levels was 64%, compared with 53% of suchidentifications when the sample was not dried between the dispenses.This indicates that the solution dispense/layering (w/ drying) methoddescribed herein provides a marked advantage over multiple dispenseswith no drying therebetween for the test of “low McFarland” samplesusing Maldi-TOF.

EXAMPLE 3

Samples (0.25 McFarland) were prepared and spiked with themicroorganisms enumerated in FIG. 3B. Samples were prepared using theprocedure described generally above. Three spots were formed on eachMaldi target plate. Three target plates were prepared for each sample.The Maldi target plates were obtained from Bruker. On the first plate,three spots of 1 μl sample each were dispensed. On the second plate,three spots of 2 μl sample each were dispensed. On the third plate,three spots of 1 μl sample each were dispensed, dried, and another 1 μlsample spot was formed on each dried spot. After the spots werecompletely deposited, the spots on the plates were dried. Then, 1 μl of70% formic acid was applied on the dried spots on both plates. Thesamples were again dried. Then 1 μl of HCCA matrix was placed over thespot and the sample was dried and analyzed on Bruker Maldi. Maldisuccessfully identified 31 out of 42 spots of spiked samples (at thegenus level) for those samples prepared with a single 1 μl dispense of0.25 McFarland sample. Maldi successfully identified 33 out of 42 spotsof spiked samples (at the genus level) for those samples prepared with asingle 2 μl dispense of 0.25 McFarland sample. Maldi successfullyidentified 36 out of 42 spots of spiked samples (at the genus level) forthose samples prepared with a 1 μl dispense of 0.25 McFarland samplefollowed by drying and a second 1 μl dispense. These results arereported in Table 3 below and in FIG. 7. When results are reported as a“best of three, it is clear that the best result (correct IDs for thegreatest number of different microorganisms) was obtained using 0.25McFarland with 1 μl/1 μl by layering.

TABLE 3 Maldi EVALUATION 0.25 McFarland 0.25 McFarland 0.25 McFarland 1μl/1 μl 1 μl Addition 2 μl Addition (Layering) Genus and 10 18 24Species ID Genus ID only 21 15 12 No ID 11  9  6 Correct ID 6/14 8/149/14 % CORRECT 24% 43% 57%

At the species level, the solution dispense/layering method using two 1μl dispenses with an intervening drying step provided 24 correctidentifications at the species level (compared with 18 for a 2 μl singledispense and 10 for a 1 μl single dispense). This data again shows thatthe solution dispense/layering method provides markedly more accurateMaldi scores than single dispenses for samples having lower McFarlandvalues.

In one embodiment, the microorganism from pure isolates or a PBC sampleis identified by: i) obtaining the sample suspected to contain at leastone microorganism from growth media (i.e. either from PBC or from asub-culture on an agar plate); ii) determining the inoculationparameters of the sample, those parameters including, for the determinedMcFarland value of the inoculate, the dispense volume and the number ofdispenses; iii) depositing at least a portion of the sample on a solidsurface adapted to be placed in an apparatus configured to determine theidentity of microorganisms by Maldi mass spectrometry using theprescribed dispense volume and number of dispenses); iv) treating thesample with at least one reagent; such reagents including a volatileacid, an organic solvent, and/or a combination of organic solvent and avolatile acid; v) placing a Maldi matrix solution over the treatedsample; and vi) identifying the microorganism by mass spectrometry. Inone embodiment the volatile acid is at least 70% formic acid. In anotherembodiment the volatile acid is at least 80% formic acid. In anotherembodiment the volatile acid is at least 90% formic acid. Unlessotherwise specified herein, the formic acid solutions are aqueoussolutions. In another embodiment the volatile acid is at least 100%formic acid (e.g. neat). In another embodiment, the sample is treatedwith at least 70% formic acid in an organic solvent such asacetonitrile, methanol, ethanol, acetone, or ethyl acetate prior toplacing a Maldi matrix solution over the sample. In another embodiment,the sample is treated with at least 80% formic acid in an organicsolvent such as acetonitrile, methanol, ethanol, acetone, or ethylacetate prior to placing a Maldi matrix solution over the sample. Inanother embodiment, the sample is treated with at least 90% formic acidin an organic solvent such as acetonitrile, methanol, ethanol, acetone,or ethyl acetate prior to placing a Maldi matrix solution over thesample. In one embodiment, the sample deposited on the solid surface isallowed to dry prior to adding the volatile acid, to prevent the samplefrom diluting the volatile acid. In another embodiment, the volatileacid is dried prior to placing the Maldi matrix solution over thesample. Examples of the volatile acids that may be used in the variousembodiments of the invention include, but are not limited to, formicacid, acetic acid, trifluoracetic acid and hydrochloric acid.

After the sample is dried on the Maldi plate and combined with thevolatile acid alone or in combination with an organic solvent, thecombination of sample and reagents is dried. Drying is defined asallowing the liquid to evaporate sufficiently so as not to dilute anyliquid subsequently added. While the sample can be dried in ambient air,a heating source, such as a heating block, hot plate, heating oven orinfrared heating lamp can be used to accelerate the evaporation of theliquid portion of the combined sample and reagents. These drying methodsdo not change the spectrum of the sample upon identification by Maldi.

After drying, the Maldi matrix is applied over the sample treated withreagent(s). Any Maldi matrix solution known to those skilled in the artcan be used in the disclosed methods. These matrix solutions include,but are not limited to, α-cyano-4-hydroxycinnamic acid (HCCA),2,5-dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid(SPA), 3-hydroxypicolinic acid (HPA), 3.4-dihydroxycinnamic acid,2-(4-hydroxyphenylazo)-benzoic acid, 2-amino-4-methyl-5-nitropyridine,and 2,4,6-trihydroxy acetophenone (THAP).

In an alternative embodiment, the microorganism is identified by: i)preparing a microbial suspension of a sample suspected of containing atleast one microorganism; ii) determining the turbidity of the microbialsuspension; iii) selecting the volume and number of dispenses to be usedto deposit, from a microbial suspension, at least a portion of thesample on a solid surface adapted to be placed in an apparatusconfigured to determine the identity of microorganisms by Maldi massspectrometry; iv) depositing the sample on the Maldi plate using theselected parameters; v) optionally, fixing the microorganism with anorganic solvent, e.g. ethanol, a fixative, e.g. formaldehyde, or byapplying heat, generally up to about 37° C. (i.e. approximately bodytemperature); vi) covering the sample with at least 70% formic acid; v)drying the sample; vi) placing a Maldi matrix solution over the treatedsample; vii) drying the sample; and viii) identifying the microorganismby mass spectrometry. Fixatives such as formaldehyde are well known toone skilled in the art and are not described in detail herein.

Referring to FIG. 8, on example of the present invention contemplatesobtaining a biological sample. The sample is combined with a diluentsuited to deliver the sample to a Maldi plate. The turbidity of thesample/diluent mixture is measured. If the measured turbidity is withina predetermined range, an aliquot with a predetermined volume isdeposited on the Maldi plate. If the measured turbidity is higher than apredetermined range, then a smaller volume of sample is deposited on theMaldi plate. If the measured turbidity is less than the predeterminedrange, then the above-described sample preparation protocol usingmultiple dispenses with drying between dispenses is used.

In one exemplary embodiment, a sample is obtained and a suspension isprepared. The turbidity is measured. If the turbidity (in McFarland) isbetween about 2 and about 6, about 3 μl is deposited on the Maldi plate.If the sample turbidity is higher than about 6, then the amount ofsample deposited on the Maldi plate is reduced to about 1 μl. If thesample turbidity is less than about 2 but in the range of about 1 toabout 2, then about 3 μl of sample is deposited on the Maldi plate,dried, and a second 3 μl sample is deposited and dried. If the sampleturbidity is about 0.5 to about 1, then three “layers” of sample, eachabout 3 μl, are deposited and dried. If the sample turbidity is about0.25 to about 0.5, then 4 “layers” of sample (3 μl each) are depositedand dried.

After the sample is deposited and dried, the samples are processed forMaldi as described herein.

An automated system for preparing a biological sample for evaluation byMatrix-Assisted Laser Desorption Ionization Time of Flight massspectrometry is also contemplated. Such system has a programmablecontroller (e.g. a processor) in communication with a sample preparationdevice. The programmable controller communicates instructions to thesample preparation device to pick a colony from a plated culture. Anexample of such a device is the InoquIA™ which is obtained commerciallyfrom BD Kiestra™. The processor provides instructions to add an amountof diluent or sample processing reagents, as appropriate, to the sampleto provide a sample solution having a predetermined volume. Automatedsystems that dilute samples to a predetermined volume are well known tothe skilled person and not described in detail herein. After sampledilution, the turbidity of the sample is measured. Turbidity is measuredby a variety of techniques including absorbance, transmittance andmanual methods. Embodiments of the system contemplate the use of anephelometer to measure turbidity.

As used herein, “nephelometer” is an instrument that is capable ofmeasuring the amount of solid particles in a suspension. As used herein,“nephelometry” refers to a method by which the amount of suspendedsolids in a suspension can be measured. Nephelometers are well known tothe skilled person and not described in detail herein.

The processor is in communication with the instrument that measuressample turbidity. The processor has a memory that stores a value orrange of value for an acceptable turbidity. The processor compares themeasure value with stored values. If the sample concentration is withinthe predetermined range, the processor provides instruction to apply afirst predetermined volume of sample to a substrate adapted to deliver asample to a device configured to perform mass spectrometry as describedabove.

If the processor determines that a measured sample concentration ishigher than the predetermined range, the processor provides instructionsto apply a second predetermined volume of sample to the substrateadapted to deliver the sample to the device configured to perform massspectrometry. The second predetermined volume is less than the firstpredetermined volume.

If the processor determines that a measured sample concentration islower than the predetermined range the processor provides instructionsto: i) apply a third predetermined volume of the sample to a substrateadapted to deliver the sample to the device configured to perform massspectrometry; ii) dry the sample; iii) apply a second application of thethird predetermined volume of the sample over the dried sample on thesubstrate; and iv) dry the sample according to the methods describedherein.

Thereafter, the processor instructs the system to apply a matrix overthe deposited sample, the matrix being adapted for use in the massspectrometer. The system also has a transport mechanism for placing theprepared sample in a mass spectrometry device for testing.

FIG. 9 schematically shows an embodiment of a system 1 for the automaticpreparation of a suspension of a sample of a microorganism according tothe invention. Said system 1 comprises a stage 2 for a culture dish 3comprising a microorganism 4 on a nutritional layer 5, such as a layerof agar gel.

The system 1 has a first picking tool 6 and a further picking tool 7. Apositioning device 8 comprises a picking tool holder 9 for, in the shownembodiment releasably holding a picking tool, in the embodiment shown inFIG. 1 the picking tool holder 9 holds the first picking tool 6. Thepositioning device 8 is arranged for positioning the first picking tool6 in a starting position (shown in solid lines in FIG. 9) above theculture dish 3 and is arranged for automatically lowering and raisingthe first picking tool 6 towards and away from the culture dish 3, suchthat the first picking tool 6 can be positioned in a position (indicatedwith broken lines 6′) in which it contacts the microorganism 4 and picksup a sample of the microorganism. After the first picking tool 6 haspicked up a sample, the positioning device 8 raises and positions thefirst picking tool 6 in a transfer position. In the embodiment shown inFIG. 9 the transfer position is identical to the starting position. Inother embodiments the starting and transfer positions may be differentfrom each other.

The system 1 according to the invention further comprises a suspensiontube holder 10 for holding a suspension tube 11 which can contain asuspension medium which is dispensed from an automatic suspension mediumdispenser 12, which in the shown embodiment has a dispensing nozzle 13for automatically dispensing a suspension medium 14 in the suspensiontube 11 held in the suspension tube holder 10. In the present embodimentthe suspension tube holder 10 is a rotatable suspension tube holder forrotating the suspension tube 11 around a vertical axis A.

A transferring device 15 is incorporated in the system 1 forautomatically transferring a picking tool from the transfer position ofthe positioning device 8 to a position above the suspension tube 11 heldin the suspension tube holder 10. In the embodiment shown, thetransferring device 15 comprises a transfer holder 16 with a graspingtool for releasably holding a picking tool. The transferring device 15is illustrated as deploying a transfer track 18, such as a rail, forlinear movement thereon as indicated by the arrows. However, other deckmounted transfer mechanisms are contemplated. In this manner thetransferring device 15 may be moved to the positioning device 8, suchthat the grasping tool 17 can take over the picking tool from thepositioning device 8. The picking tool holder 9 thereafter releases thepicking tool after the grasping means 17 has grasped the picking tool.In the embodiment shown in FIG. 9 the second or further picking tool 7having previously picked up a sample 19 of the microorganism 4 ispositioned above the suspension tube 11 by the transferring device 15 ina starting position indicated by solid lines. The transferring device 15is arranged for lowering the second picking tool 7 into the suspensionmedium 14 contained in the suspension tube 11, in which position thesecond picking tool 7′ with the sample 19 is submerged in the suspensionmedium 14 as indicated by broken lines in FIG. 9. Thereafter thetransferring device 15 positions the second picking tool 7 in a waitingposition above the suspension tube 11, which waiting position is in theembodiment shown in FIG. 9 identical to the starting position. In otherembodiments the waiting position and the starting position may bedifferent from each other.

The system further is provided with a turbidity meter 20 for performingmeasurements of the turbidity of the suspension medium 14 contained inthe suspension tube 11 held in the suspension tube holder 10. Asgenerally known in the art a turbidity meter can provide measurementvalues which are a measure of the concentration of material, in thepresent case the concentration of a microorganism suspended in thesuspension medium. In the embodiment shown in FIG. 9 the turbidity meter20 comprises a laser 21 which transmits laser light towards and throughthe suspension medium and a sensor 22 which detects the amount of laserlight transmitted through the suspension medium. In addition there is afurther sensor (not indicated in the drawing) which is e.g. arrangedperpendicular to the path of the laser light to detect the amount oflaser light which has been scattered by the suspension.

The operation of the inventive device is controlled by a controller 23,for example comprising a microprocessor, which is communicativelyconnected (as indicated by the signal lines) to the positioning device8, the transferring device 15, the automatic suspension medium dispenser12, and the turbidity meter 20 for automatically controlling themovement of the positioning device 8, the movement of the transferringdevice 15, the operation of the automatic suspension medium dispenser 12and the operation of the turbidity meter 20, respectively. In additionthe controller 23 might be directly communicatively connected to otherparts of the system 1 such as, for example, the pick tool holder 9, thetransfer holder 16, the laser 21 and the sensor 22.

In the embodiment shown in FIG. 9 the controller 23 is arranged forcontrolling the turbidity meter 20 such that the turbidity measurementof the suspension medium 14 is started before the second picking tool 7is submerged in the suspension medium 14. In addition, the controller 23controls the rotatable suspension tube holder 10 for starting therotation of the suspension tube 11 held in the holder 10 before thesecond picking tool 7 is submerged in the suspension medium 14, and formaintaining the rotation of the suspension tube 11 during themeasurement of the turbidity of the suspension medium 14. In this mannerthe turbidity meter 20 provides an on-line measurement value to thecontroller 23 which value is indicative of the measured turbidity, andthus the concentration of the microorganism.

The controller 23 comprises a memory, in which a first and a secondthreshold value are stored, in which the first threshold value is equalto or greater than the second threshold value. If the turbiditymeasurement value provided by the turbidity meter is equal to or betweenthe first and second threshold value, the concentration/amount ofmicroorganism in the suspension medium is sufficient to allow thesuspension tube with the suspension to be further processed. In thatcase the controller 23 provides a signal that the suspension tube can beprocessed further. In addition in this situation the second picking tool7 can be discarded e.g. by transferring the transferring device to aposition C in which the grasping means 17 are activated to release thesecond picking tool 7.

In case the final measurement value of the turbidity meter is above thefirst threshold value previously stored in a memory of the controller 23then the concentration of the microorganism is too high to allow thesuspension tube to be processed further. In that situation thecontroller 23 controls the automatic suspension medium dispenser 12 tosupply an additional amount of suspension medium into the suspensiontube 11. This additional amount of suspension medium is based on theinitial amount of suspension medium, the final measurement value and thevalue of the first and/or second threshold value such that the additionof the additional amount of suspension medium to the suspension mediumalready present in the suspension tube 11 will lead to a concentrationof microorganism in the suspension medium which satisfies therequirement for further processing, as can be confirmed by an additionalor further measurement of the turbidity by the turbidity meter 20.

In case the final measurement value of the turbidity meter 20 is belowthe second threshold value, meaning that the concentration ofmicroorganism in the suspension medium is too low, the controller 23controls the system 1 such that an additional sample of microorganism ispicked up by the first picking tool 6 (alternatively the second oranother picking tool can be used for picking up an additional sample).In yet other embodiments (described hereinbelow), multiple dispenses ofsuspension can be deposited in the same spot if the turbidity of thesuspension is below specification. Thus, the controller 23 in this casecontrols the positioning of the transfer device 15 such that the secondpicking tool 7 is discarded as described above. Then (or simultaneously)the first picking tool 6 in the picking tool holder 9 of the positioningdevice 8 is lowered from the starting position above the culture dish 3towards the culture dish and into contact with the microorganism 4 topick up an additional sample of the microorganism. Thereafter, the firstpicking tool 6 is automatically raised with the additional sample of themicroorganism away from the culture dish to the transfer position. Thenthe transferring device automatically transfers the first picking toolwith the additional sample of the microorganism from the transferposition of the positioning device 8 to a position above the suspensiontube 11. The first picking tool 6 with the additional sample of themicroorganism is lowered into the suspension medium 14 and releases theadditional sample of the microorganism in the suspension medium. Againthe turbidity is measured, and the measured value is compared with thefirst and second threshold value stored in the memory of the controller23. In this case the controller 23 can be arranged for controlling themovement of the transferring device 15 such that the first picking tool6 is raised to the waiting position if the on-line measurement value ofthe turbidity performed by the turbidity meter 20 is equal to or lowerthan the first threshold value and equal to or greater than the secondthreshold value.

Suspension tubes, or alternately, vials or cuvettes which areparticularly useful in the inventive system have a cross-section with atarget maximum dimension of about 2 to about 12 mm, preferably about 3mm. In these relatively small suspension tubes the controller 23 cancontrol the automatic suspension medium dispenser 12 such that thesupplied initial amount of suspension medium is about 0.1-5 ml,preferably less than about 1 ml.

After the suspension is prepared, aliquots of the suspension areautomatically pipetted from the suspension tube 11 and deposited ontothe Maldi plate 100. Maldi plate 100 is schematically illustrated asresting on support 105. This step is performed at a Maldi platepreparation station identified as location C that is apart from thelocation where the suspension is prepared. In certain embodiments thesuspension tube 11 is moved to the location near the Maldi plate forMaldi plate preparation as described above. In other embodiments, analiquot of suspension is removed from suspension tube 11 and thataliquot is used for Maldi plate preparation. The robotic pipettor isillustrated schematically as 110 in FIG. 9. Robotic pipettors are wellknown and not described in detail herein. The Maldi plate 100 isconnected to a drying apparatus illustrated as heater 120.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

1. A method for preparing a biological sample for evaluation byMatrix-Assisted Laser Desorption Ionization Time of Flight massspectrometry the method comprising: obtaining a biological sample;diluting the biological sample; measuring a concentration of thebiological sample; if the measured biological sample concentration iswithin a predetermined range, applying a first predetermined aliquot ofthe biological sample to a surface adapted to deliver the appliedbiological sample to a device configured to perform mass spectrometry;if a measured biological sample concentration is higher than thepredetermined range, applying a second predetermined aliquot of thebiological sample to the surface adapted to deliver the appliedbiological sample to the device configured to perform mass spectrometry,wherein the second predetermined aliquot is less than the firstpredetermined aliquot; if a measured biological sample concentration islower than the predetermined range, i) applying a first aliquot of thebiological sample to the surface adapted to deliver the biologicalsample to the device configured to perform mass spectrometry; ii) dryingthe sample; iii) applying a second aliquot of the biological sample overthe dried sample from the first aliquot; iv) drying the biologicalsample; applying a matrix solution over the applied biological sample,the matrix solution being adapted for use in the mass spectrometer; andperforming mass spectrometry on the biological sample.
 2. The method ofclaim 1 wherein the biological sample concentration is measured by oneof absorbance, transmittance, manual or nephelometery.
 3. The method ofclaim 2 wherein the biological sample concentration is measured bynephelometry.
 4. The method of claim 1 wherein the biological sample isa portion of a colony of a microorganism picked from a culture plate. 5.The method of claim 1 wherein the biological sample is collected from apositive blood culture.
 6. The method of claim 1 wherein the surface isa plate adapted for use in a mass spectrometer.
 7. The method of claim 3wherein the predetermined range is about 2 McFarland to about 6McFarland and wherein the diluted biological sample has a turbidity ofabout 1 to less than about 2 McFarland, the first and second aliquotsare both about 3 μl.
 8. The method of claim 1 wherein the matrixsolution is selected from the group consisting ofα-cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB),3,5-dimethoxy-4-hydroxycinnamic acid (SPA), 3-hydroxypicolinic acid(HPA), 3.4-dihydroxycinnamic acid, 2-(4-hydroxyphenylazo)-benzoic acid,2-amino-4-methyl-5-nitropyridine, and 2,4,6-trihydroxy acetophenone(THAP).
 9. The method of claim 1 wherein the biological sample is driedat a temperature of about room temperature to about 45° C.
 10. Themethod of claim 1 wherein, after application of the biological sample tothe surface, the biological sample is treated with a reagent comprisingat least one of a volatile acid, an organic solvent and combinationsthereof.
 11. The method of claim 10 where in the volatile acid is atleast 70% formic acid.
 12. The method of claim 11 wherein the formicacid is combined with an organic solvent and the organic solvent isselected from the group consisting of acetonitrile, methanol, ethanol,acetone, or ethyl acetate.
 13. The method of claim 1 wherein thepredetermined range is about 2 McFarland to about 6 McFarland andwherein the diluted biological sample has a turbidity of about 0.5 toless than about 1 McFarland, the method further comprising applying afirst aliquot of about 3 μl on the surface, drying the biologicalsample, applying a second aliquot of about 3 μl on the surface, dryingthe biological sample, applying a third aliquot of about 3 μl on thebiological sample and drying the biological sample.
 14. The method ofclaim 1 wherein the predetermined range is about 2 McFarland to about 6McFarland and wherein the diluted biological sample has a turbidity ofabout 0.25 to less than about 0.5 McFarland, the method furthercomprising applying a first aliquot of about 3 μl on the surface, dryingthe biological sample, applying a second aliquot of about 3 μl on thesurface, drying the biological sample, applying a third aliquot of about3 μl on the biological sample, drying the biological sample, applying afourth aliquot of about 3 μl on the biological sample, and drying thebiological sample.
 15. An automated system for preparing a biologicalsample for evaluation by Matrix-Assisted Laser Desorption IonizationTime of Flight mass spectrometry the automated system comprising: aprocessor in communication with an automated apparatus that prepares abiological sample for an evaluation by Matrix-Assisted Laser DesorptionIonization Time of Flight mass spectrometry wherein the processor: i)provides instructions to the automated apparatus which is adapted toobtain a biological sample from a culture media; and ii) providesinstructions to the automated apparatus that combines a volume of adiluent determined by the processor with the biological sample toprovide a diluted sample; the automated apparatus comprising anephelometer that measures a concentration of the diluted sample whereinthe nephelometer communicates with the processor; wherein the processorreceives the measured concentration of the diluted sample and determinesif the measured concentration of the diluted sample is within apredetermined range, and, if the processor determines that the dilutedsample is within the predetermined range, the processor providesinstructions to the automated apparatus to apply a first predeterminedvolume of diluted sample to a substrate adapted to deliver a sample to adevice configured to perform mass spectrometry; wherein, if theprocessor determines that a measured sample concentration is higher thanthe predetermined range, the processor provides instructions to theautomated apparatus to apply a second predetermined volume of sample tothe substrate adapted to deliver the sample to the device configured toperform mass spectrometry, wherein the second predetermined volume isless than the first predetermined volume; wherein, if the processordetermines that a measured sample concentration is lower than thepredetermined range the processor provides instructions to the automatedapparatus to: i) apply a third predetermined volume of the sample to asubstrate adapted to deliver the sample to the device configured toperform mass spectrometry; ii) dry the sample; iii) apply a secondapplication of the third predetermined volume of the sample over thedried sample on the substrate; and iv) dry the sample; wherein theprocessor then instructs the automated system to apply a matrix over theapplied sample, the matrix being adapted for use in the massspectrometer; and the automated system further comprising a transportmechanism for placing the prepared sample in a mass spectrometry devicefor testing.
 16. The automated system of claim 15 comprising a heaterfor drying the sample.
 17. The automated system of claim 15 wherein thepredetermined sample concentration range of the diluted sample is about2 to about 6 McFarland.
 18. The automated system of claim 15 wherein thethird predetermined volume is about 3 μl.
 19. The automated system ofclaim 15 wherein the first predetermined volume is about 3 μl.
 20. Theautomated system of claim 15 wherein the second predetermined volume isabout 1 μl.
 21. The automated system of claim 15 wherein, if the dilutedsample has a turbidity of about 1 to less than about 2 McFarland, thediluted sample is applied to the substrate in first and secondapplications with a drying step following each application.
 22. Theautomated system of claim 15 wherein, if the diluted sample has aturbidity of about 0.5 to less than about 1 McFarland, the dilutedsample is applied to the substrate in first, second, and thirdapplications with a drying step following each application.
 23. Theautomated system of claim 15 wherein, if the diluted sample has aturbidity of about 0.25 to less than about 0.5 McFarland, the dilutedsample is applied to the substrate in first, second, third and fourthapplications with a drying step following each application.